Estimation of flow-induced deformation of glycocalyx layer on vascular endothelial cells: comparison of three methods

The endothelial glycocalyx layer plays a crucial role in mechanotransduction in response to fluid shear stress, yet its actual deformation under physiological fluid shear stress remains poorly understood. The present study aimed to quantify the shear-induced deformation of the glycocalyx on vascular endothelial cells using three independent experimental approaches. Mouse vascular endothelial cell line F-2 was cultured in a custom-made flow chamber and observed on a confocal laser scanning microscope while applying fluid shear stress. In approach I, we analyzed changes in the fluorescence intensity profile of the region between photobleached and nonphotobleached regions in fluorescently labeled glycocalyx layer induced by application of fluid shear stress, to infer shear deformation. In approach II, shear strain was estimated by measuring the reduction in layer thickness under oscillatory shear stress. In approach III, horizontal displacements of the glycocalyx layer were directly measured using quantum dots attached to the glycocalyx surface and the cell membrane subjected to fluid shear stress. Across all methods, the glycocalyx layer exhibited consistent deformation with estimated shear strain values ranging from 3° to 10° per pascal of shear stress. From these findings, the Young's modulus of the glycocalyx layer was estimated to be 16-50 Pa, markedly lower than the modulus of the plasma membrane, suggesting high deformability. These results provide the first direct quantification of glycocalyx deformation under physiological-like shear conditions and offer critical insight into its mechanosensory function. Our findings support the concept that the endothelial glycocalyx layer actively contributes to flow-induced signaling and vascular function.NEW & NOTEWORTHY Endothelial glycocalyx layer deformation under physiological fluid flow remains to be understood. This study provides the first direct quantification of shear-induced deformation of the endothelial glycocalyx layer using three distinct confocal microscopic approaches: photobleaching, thickness measurement, and quantum dots imaging. The observed shear strains from three approaches were consistent (3-10°/Pa), revealing that the glycocalyx is actually deformed in response to fluid shear stress and may serve as a key mechanosensor in vascular endothelial cells.